Nectarios Klonis

Trafficking and assembly of the cytoadherence complex in Plasmodium falciparum-infected human erythrocytes

After invading human erythrocytes, the malarial parasite Plasmodium falciparum, initiates a remarkable process of secreting proteins into the surrounding erythrocyte cytoplasm and plasma membrane. One of these exported proteins, the knob‐associated histidine‐rich protein (KAHRP), is essential for microvascular sequestration, a strategy whereby infected red cells adhere via knob structures to capillary walls and thus avoid being eliminated by the spleen. This cytoadherence is an important factor in many of the deaths caused by malaria. Green fluorescent protein fusions and fluorescence recovery after photobleaching were used to follow the pathway of KAHRP deployment from the parasite endomembrane system into an intermediate depot between parasite and host, then onwards to the erythrocyte cytoplasm and eventually into knobs. Sequence elements essential to individual steps in the pathway are defined and we show that parasite‐derived structures, known as Maurers clefts, are an elaboration of the canonical secretory pathway that is transposed outside the parasite into the host cell, the first example of its kind in eukaryotic biology.

Artemisinin activity against Plasmodium falciparum requires hemoglobin uptake and digestion

Combination regimens that include artemisinin derivatives are recommended as first line antimalarials in most countries where malaria is endemic. However, the mechanism of action of artemisinin is not fully understood and the usefulness of this drug class is threatened by reports of decreased parasite sensitivity. We treated Plasmodium falciparum for periods of a few hours to mimic clinical exposure to the short half-life artemisinins. We found that drug treatment retards parasite growth and inhibits uptake of hemoglobin, even at sublethal concentrations. We show that potent artemisinin activity is dependent on hemoglobin digestion by the parasite. Inhibition of hemoglobinase activity with cysteine protease inhibitors, knockout of the cysteine protease falcipain-2 by gene deletion, or direct deprivation of host cell lysate, significantly decreases artemisinin sensitivity. Hemoglobin digestion is also required for artemisinin-induced exacerbation of oxidative stress in the parasite cytoplasm. Arrest of hemoglobin digestion by early stage parasites provides a mechanism for surviving short-term artemisinin exposure. These insights will help in the design of new drugs and new treatment strategies to circumvent drug resistance.

Spectral properties of the prototropic forms of fluorescein in aqueous solution

The commonly used fluorescent probe, fluorescein, can exist in seven prototropic forms. We have used global analysis procedures to reanalyze the absorption data of Diehl and Horchak-Morris (Talanta34, 739–741, 1987) in terms of five alternative ionization models. We identify the forms of fluorescein present in aqueous solution and the pKa of each ionisation transition. The pKa values of the neutral xanthene, carboxylic acid, and cationic xanthene groups are 6.3, 3.1–3.4, and 3.1–3.4, respectively, and the pKa value of lactonization is 2.4. As a consequence, the neutral form of fluorescein is a mixture of the lactone (70%), zwitterionic (15%), and quinoid (15%) forms. A knowledge of the forms present in solution permits the characterization of their spectral properties. It is shown that the quinoid and monoanion forms have similar absorption spectra, while the zwitterion spectrum is similar to that of the cation but blue-shifted by 3 nm. The emission spectra of the monoanion and quinoid forms are also identified and shown to be similar but not identical. A model for the excited-state reactions of fluorescein is presented.

Food vacuole‐associated lipid bodies and heterogeneous lipid environments in the malaria parasite, Plasmodium falciparum

The malaria parasite Plasmodium falciparum induces a sixfold increase in the phospholipid content of infected erythrocytes during its intraerythrocytic growth. We have characterized the lipid environments in parasitized erythrocyte using the hydrophobic probe, Nile Red. Spectral imaging with a confocal microscope revealed heterogeneous lipid environments in parasite‐infected erythrocytes. An insight into the nature of these environments was gained by comparing these spectra with those of triacylglycerol/phospholipid emulsions and phospholipid membranes. Using this approach, we identified a population of intensely stained particles of a few hundred nanometers in size that are closely associated with the digestive vacuole of the parasite and appear to be composed of neutral lipids. Electron microscopy and isolation of food vacuoles confirmed the size of these particles and their intimate association respectively. Lipid analysis suggests that these neutral lipid bodies are composed of di‐ and triacylgycerols and may represent storage organelles for lipid intermediates that are generated during digestion of phospholipids in the food vacuole. Mono‐, di‐ and triacylglycerol suspensions promote β‐haematin formation, suggesting that these neutral lipid bodies, or their precursors, may also be involved in haem detoxification. We also characterized other compartments of the infected erythrocyte that were stained less intensely with the Nile Red probe. Both the erythrocyte membrane and the parasite membrane network exhibit red shifts compared with the neutral lipid bodies that are consistent with cholesterol‐rich and cholesterol‐poor membranes respectively. Ratiometric imaging revealed more subtle variations in the lipid environments within the parasite membrane network.

Fluorescence photobleaching analysis for the study of cellular dynamics

Abstract The wide availability of the confocal microscope and the emergence of green fluorescent protein (GFP) transfection technology has led to the increasing use of photobleaching studies to examine aspects of cellular dynamics in living cells. In this review, we examine the theory and practice of performing photobleaching studies using a confocal microscope. We illustrate the application of photobleaching protocols using our own measurements of fluorescently labelled red blood cells and of malaria parasite-infected erythrocytes expressing GFP fusions and examine other examples from the literature.

Genesis of and trafficking to the Maurer's clefts of Plasmodium falciparum-infected erythrocytes†

ABSTRACT Malaria parasites export proteins beyond their own plasma membrane to locations in the red blood cells in which they reside. Maurers clefts are parasite-derived structures within the host cell cytoplasm that are thought to function as a sorting compartment between the parasite and the erythrocyte membrane. However, the genesis of this compartment and the signals directing proteins to the Maurers clefts are not known. We have generated Plasmodium falciparum-infected erythrocytes expressing green fluorescent protein (GFP) chimeras of a Maurers cleft resident protein, the membrane-associated histidine-rich protein 1 (MAHRP1). Chimeras of full-length MAHRP1 or fragments containing part of the N-terminal domain and the transmembrane domain are successfully delivered to Maurers clefts. Other fragments remain trapped within the parasite. Fluorescence photobleaching and time-lapse imaging techniques indicate that MAHRP1-GFP is initially trafficked to isolated subdomains in the parasitophorous vacuole membrane that appear to represent nascent Maurers clefts. The data suggest that the Maurers clefts bud from the parasitophorous vacuole membrane and diffuse within the erythrocyte cytoplasm before taking up residence at the cell periphery.

Targeting the Cell Stress Response of Plasmodium falciparum to Overcome Artemisinin Resistance

Successful control of falciparum malaria depends greatly on treatment with artemisinin combination therapies. Thus, reports that resistance to artemisinins (ARTs) has emerged, and that the prevalence of this resistance is increasing, are alarming. ART resistance has recently been linked to mutations in the K13 propeller protein. We undertook a detailed kinetic analysis of the drug responses of K13 wild-type and mutant isolates of Plasmodium falciparum sourced from a region in Cambodia (Pailin). We demonstrate that ART treatment induces growth retardation and an accumulation of ubiquitinated proteins, indicative of a cellular stress response that engages the ubiquitin/proteasome system. We show that resistant parasites exhibit lower levels of ubiquitinated proteins and delayed onset of cell death, indicating an enhanced cell stress response. We found that the stress response can be targeted by inhibiting the proteasome. Accordingly, clinically used proteasome inhibitors strongly synergize ART activity against both sensitive and resistant parasites, including isogenic lines expressing mutant or wild-type K13. Synergy is also observed against Plasmodium berghei in vivo. We developed a detailed model of parasite responses that enables us to infer, for the first time, in vivo parasite clearance profiles from in vitro assessments of ART sensitivity. We provide evidence that the clinical marker of resistance (delayed parasite clearance) is an indirect measure of drug efficacy because of the persistence of unviable parasites with unchanged morphology in the circulation, and we suggest alternative approaches for the direct measurement of viability. Our model predicts that extending current three-day ART treatment courses to four days, or splitting the doses, will efficiently clear resistant parasite infections. This work provides a rationale for improving the detection of ART resistance in the field and for treatment strategies that can be employed in areas with ART resistance.

Altered temporal response of malaria parasites determines differential sensitivity to artemisinin

Reports of emerging resistance to first-line artemisinin antimalarials make it critical to define resistance mechanisms and identify in vitro correlates of resistance. Here we combine unique in vitro experimental and analytical approaches to mimic in vivo drug exposure in an effort to provide insight into mechanisms of drug resistance. Tightly synchronized parasites exposed to short drug pulses exhibit large stage-dependent differences in their drug response that correlate with hemoglobin digestion throughout most of the asexual cycle. As a result, ring-stage parasites can exhibit >100-fold lower sensitivity to short drug pulses than trophozoites, although we identify a subpopulation of rings (2–4 h postinvasion) that exhibits hypersensitivity. We find that laboratory strains that show little differences in drug sensitivity in standard in vitro assays exhibit substantial (>95-fold) difference in sensitivity when exposed to short drug pulses. These stage- and strain-dependent differences in drug sensitivity reflect differential response lag times with rings exhibiting lag times of up to 4 h. A simple model that assumes that the parasite experiences a saturable effective drug dose describes the complex dependence of parasite viability on both drug concentration and exposure time and is used to demonstrate that small changes in the parasite’s drug response profile can dramatically alter the sensitivity to artemisinins. This work demonstrates that effective resistance can arise from the interplay between the short in vivo half-life of the drug and the stage-specific lag time and provides the framework for understanding the mechanisms of drug action and parasite resistance.

Digestive-vacuole genesis and endocytic processes in the early intraerythrocytic stages of Plasmodium falciparum.

The digestive vacuole of the malaria parasite Plasmodium falciparum is the site of haemoglobin digestion and haem detoxification, and is the target of chloroquine and other antimalarials. The mechanisms for genesis of the digestive vacuole and transfer of haemoglobin from the host cytoplasm are still debated. Here, we use live-cell imaging and photobleaching to monitor the uptake of the pH-sensitive fluorescent tracer SNARF-1-dextran from the erythrocyte cytoplasm in ring-stage and trophozoite-stage parasites. We compare these results with electron tomography of serial sections of parasites at different stages of growth. We show that uptake of erythrocyte cytoplasm is initiated in mid-ring-stage parasites. The host cytoplasm is internalised via cytostome-derived invaginations and concentrated into several acidified peripheral structures. Haemoglobin digestion and haemozoin formation take place in these vesicles. The ring-stage parasites can adopt a deeply invaginated cup shape but do not take up haemoglobin via macropinocytosis. As the parasite matures, the haemozoin-containing compartments coalesce to form a single acidic digestive vacuole that is fed by haemoglobin-containing vesicles. There is also evidence for haemoglobin degradation in compartments outside the digestive vacuole. The work has implications for the stage specificity of quinoline and endoperoxide antimalarials.

Electron tomography of the Maurer's cleft organelles of Plasmodium falciparum-infected erythrocytes reveals novel structural features.

During intraerythrocytic development, the human malaria parasite, Plasmodium falciparum, establishes membrane‐bound compartments, known as Maurers clefts, outside the confines of its own plasma membrane. The Maurers compartments are thought to be a crucial component of the machinery for protein sorting and trafficking; however, their ultrastructure is only partly defined. We have used electron tomography to image Maurers clefts of 3D7 strain parasites. The compartments are revealed as flattened structures with a translucent lumen and a more electron‐dense coat. They display a complex and convoluted morphology, and some regions are modified with surface nodules, each with a circular cross‐section of ∼25 nm. Individual 25 nm vesicle‐like structures are also seen in the erythrocyte cytoplasm and associated with the red blood cell membrane. The Maurers clefts are connected to the red blood cell membrane by regions with extended stalk‐like profiles. Immunogold labelling with specific antibodies confirms differential labelling of the Maurers clefts and the parasitophorous vacuole and erythrocyte membranes. Spot fluorescence photobleaching was used to demonstrate the absence of a lipid continuum between the Maurers clefts and parasite membranes and the host plasma membrane.